这项工作是由在NHLBI刻录赠款支持。

1。电力塔的安装和操作<ol><li>电力塔被放置在一块长方形的胶合板被钳制表。 4.5齿轮电机转速从格兰杰在于四个2“5233”板和胶合板1¾英寸之上。电机，交流/直流调速，保险丝，开关/连接在一起到契机框。 </li><li>冷却电机，切成一个洞直径3英寸的垂直半英寸的一块胶合板。一方面，一个3英寸的风扇连接到胶合板，另一方面，3英寸直径的金属圆柱连接。 </li><li>一个特制的旋转手臂上月底安装滚筒连接到电机。电机和连接手臂以顺时针方向旋转，推向下弯曲¾英寸的方形管股票，与门的铰链连接到胶合板的一面。车轮连接到管库存低于平台的顶面，使其能够推出针对的平台，是DIRectly以上。 </li><li>手臂推管股票的一端，举起另一端，提高两级平台（图1D）。当手臂上辊清除管股票，它可以让手臂回到起始位置，放弃平台回落（图1F）。 </li><li>该平台包括胶合板上，并迷上了钉眼上的绳索固定放置的机架。该平台上下滑动沿抽屉滑块连接到胶合板用括号。 </li><li>样品瓶放置到机架上，并获得由方格屏幕，在绳索的地方举行。 </li><li>一个泡沫塑料垫放在下面的平台，以抑制下降的冲击。 </li><li>电机功率调整这样一个完整的自转周期是15秒之久。 </li></ol> 2。演习协议<ol><li>苍蝇被安置在25℃孵化湿度50％和12小时的光/ DARK周期。在25°C的温度控制室内进行的运动训练和运动能力的评估。 </li><li>苍蝇收集和年龄相匹配的，对方在1-2天。至少需要耐力和的负面geotaxis（攀岩）的能力，两个反映运动训练效果的生理指标的纵向监测的240苍蝇。纵向监测，可以为每个队列的初始值的相对变化诱导的隔离，没有与同伙变化的影响因子。应收集需要额外的实验中的苍蝇。 </li><li>采集后，苍蝇分为120实验的苍蝇，这将受到运动处方，120个控制苍蝇，这将不获行使。苍蝇苍蝇每个存储20瓶。实验苍蝇的小瓶插入苍蝇软SPO插入公司纤维素醋酸Flugs的控制瓶（未行使）的NGE瓶塞，可以下推固定，而在电力塔上的苍蝇。海绵用于固定，因为它的自适应大小，允许容易放置小瓶底部附近。柔软的海绵材料也减少了更加坚实的Flugs所造成的伤害。 </li><li>电力塔上的苍蝇都存储在小瓶含有5毫升的食品，它提供了比空药瓶行使的苍蝇软着陆行使。食物通常是由蔗糖10％，10％的酵母，水和2％琼脂，虽然我们没有观察苍蝇在一堂训练课的过程中经常吃。如果在训练过程中使用的空药瓶，遭受伤害的苍蝇的比例大大增加。 </li><li>电力塔操作温度控制的房间保持在25°C。实验苍蝇放在电力塔和攀登。控制苍蝇也被放置在电塔，但海绵塞暴射d往下到小瓶约3毫米以上的苍蝇，以限制运动。这个小组作为一个控制电力塔无关行使任何影响。虽然这些苍蝇仍然存在一些空间机动，我们已多次指出，苍蝇在这些条件下运​​行的很少，没有展示出一种生理反应，以行使。这也可能是可取的，包括一个额外的控制组，不放置在电力塔上的。 </li><li>苍蝇行使连续5天每星期利用一个倾斜的时间表（图2）。苍蝇在本周一的行使为2.5小时，每节2小时，每节，本周二，每周3小时，每节三。虽然已经过测试，其他方案，这个协议产生不同基因型和不同条件下的一致的结果。然而，该协议是受到多种可能的变化，可能适合一个特定的实验。 </li></ol><p=“jove_title”的3课。运动和运动疲劳<ol><li>在耐力运动疲劳的时间，以确认和/或定量某一世代的运动能力，是一个有益的生理指标。纵向上同伙将用于培训实验，可以进行疲劳检测。检测可进行训练之前，完成后的培训协议，或两者兼而有之。可以添加其他中间的时间点，如果图表循序渐进的训练期间所需。疲劳试验，也可以用来衡量运动耐力，比培训协议，如饮食习惯的改变（图3B），其他治疗的反应。 </li><li>每天训练时不发生，应进行疲劳检测。两个实验组和对照苍蝇放在电力塔，每组20瓶和爬直到疲倦。登山是回应负geotaxis刺激未能被确认为疲劳havior。目测取得疲劳行为。 </li><li>一小瓶的苍蝇被认为是“审美疲劳”时，5个或更少的苍蝇是能够连续四年下降爬上高于2英寸。疲劳小瓶从电塔被删除，而它仍然经营，搬迁的时间记录。搬迁的时间可以绘制作为嵌套直方图，或作为“故障时间”的阴谋。例如数据绘制成“故障时间”的情节，如图3B所示。 </li><li>样品瓶监测最多为10小时10分钟的时间间隔（图3B）。 </li></ol> 4。锻炼，年龄和运动能力<ol><li>由于本次培训协议依赖引起的运行行为，主要检测了定量的训练效果是纵向衡量在培训期间的平均运行速度的变化。在培训过程中，行使及未行使的苍蝇120飞的同伙，每天测试他们的反应负geotaxis小号timulus。环技术加尔加诺等。 （2005）是用于测量果蝇的爬坡能力12。简单地说，这一技术措施，平均身高一小瓶苍蝇爬上定义期间诱导负geotaxis后的时间。在规定的时间攀升的高度相当于爬的速度。测量之前进行日常训练，以避免疲劳一天的运行后并发症的机器上放置苍蝇。 </li><li>苍蝇在我们的标准协议，行使共有三个星期，爬坡能力强，为五周总：三个星期后停止运动，在运动训练两个星期检查（图2）。这允许实验者图表是否行使对流动性的影响后，坚持戒烟的计划。 </li><li>每个队列的日常环性能的四个照片用于爬坡能力分析。六瓶是包含在每一张照片。负Geotaxis被诱导，并采用定时相机，照片拍摄后几秒钟。我们通常使用两秒钟，虽然这取决于实验条件，可以是多种多样的。对于每一张照片，每小瓶分为四个象限的高度相等，是用来给每个飞得分的基础上象限，它在规定的时间内达到和图像处理软件。爬上顶层象限得到4分，苍蝇飞在未来的最高象限接收3，飞行中的第二个最高象限获得2，苍蝇在最低的象限接收1。苍蝇不爬在所有的底部给出了0分。 </li><li>对于每一个给定的照片小瓶，“攀登指数”所产生的小瓶中，所有果蝇的平均分数。然后平均每个小瓶的指数从四张照片给了这一天的最后攀登指数。 </li><li>所有负geotaxis实验进行纵dinally。为了减少队列变化的影响，在爬坡能力和突出的世代的攀岩运动训练的结果的能力相对纵向变化的结果归到最初的“运动前”得分。初始的前三天的登山指数的平均得分是由被分配一个值1。随后的审判，然后作为初始指数的百分比表示。 </li></ol> 5。代表结果野生型果蝇响应与减少与年龄有关的下降耐力协议，爬坡能力强，仍然存在培训结束后，体现在五个星期的年龄（图3A）纵向环检测。推迟这种负geotaxis的下降是一个标准的表型反应，可作为阳性对照，以确保野生型的运动响应通常发生。这个数据集作为一个例子是如何提出的作为行为的输入，可用于诱导行使电力塔计划。然后可以评估各种遗传或环境因素，以调节这种影响的能力。 相反，电塔也可以被用来作为各种实验设计的输出。例如，基因型，饮食，或其他条件的，可以是多种多样的。然后可以测试使用的电力塔，运动生理学上的这些变化的影响。在这里，我们表明这种方法的一个例子。当与不同比例的蔗糖在他们的饮食飞行疲劳，蔗糖含量增加，增加续航能力（图3B）相关的时间进行了测试。 <img alt="图1" src="/files/ftp_upload/3786/3786fig1.jpg" /> 图1。电力塔的操作。 （AC）电动带有附加辊弯曲手臂顺时针方向旋转，直到它接触到的弯曲方管（D和E）。手臂弯曲方管上推下来，造成瓶苍蝇解除载货平台（F）由于手臂清除管平台允许向下回落，迫使苍蝇返回底部小瓶。 <img alt="图2" src="/files/ftp_upload/3786/3786fig2.jpg" /> 图2。建议运动议定书 。苍蝇受到培训协议是由三个星期长的斜坡方案，逐步增加，从最初的2个小时的运动时间由30分钟，每星期，每星期锻炼五天。标准的分析，包括疲劳检测前和锻炼计划，并从1至5周周环检测。作为阴性对照未行使苍蝇数目相等，所有的实验都重复进行。研究员确定的，可以进行其他各种生理或生化检验。 <img aLT =“图3”的src =“/ files/ftp_upload/3786/3786fig3.jpg”/&gt; 图3。耐力运动改变流动性的多个方面。（一）环实验进行跨年龄的纵向单对一个男性的Y 1瓦67C23同伙。年龄相匹配的基因完全相同的行使及尚未行使控制苍蝇每天平均登山速度。结果均表示在一个登山指数，整个第3天测量到的平均爬升高度正常化方面。运动训练苍蝇显示在负面geotaxis能力减少与年龄有关的下降相比，跨越年龄（2路ANOVA，P &lt;0.005），年龄相匹配的未行使的兄弟姐妹。 （二）进行8小时的疲劳检测年龄匹配的女性广小号苍蝇上显示的时间疲劳（对数秩，P &lt;0.0001）的膳食蔗糖含量显着效果。实验之前，苍蝇饲喂10％重量/体积酵母/蔗糖/琼脂饮食，是ST浓度，不同比例的膳食蔗糖。五，每小瓶20苍蝇进行为每个饮食。图形显示多少瓶仍运行在一个给定的时间点有5个或更多的苍蝇。这些结果可以作为一种生存曲线（或无故障时间）与“失败”被定义为一个时间点时，不到五年的苍蝇继续以应对负geotaxis刺激一小瓶，统计学处理和图形。请注意，许多其他可能的研究设计和统计处理，数据处理和测量应量身定做适合个人的目的。

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我什么都没有透露。

这里一般协议，已成功地记录训练后生理效应。然而，在这个协议中的几个方面是受修改，以适应特定的实验需要。例如，培训的长度和数量的较量可能将被修改，以使程序或多或少具有挑战性的，需要。可以改变负geotaxis能力测量容器的高度，以增加可用面积为改善记录。也可能是适用各种自动化攀爬速度的定量方法。可用于任何软件程序能够区别于背景苍蝇的原则，加快数据收集过程。 应只修改该协议的某些方面十分谨慎，但是。例如，初步实验表明，至少每周休息一天，往往以促进更大的展示次数ovement比无情的日常锻炼。此外，昼夜节律和温度被称为影响冷血动物运动的。一天的时间，培训发生可以多种多样，但应该总是在特定群体比较一致下，为了避免昼夜节律的影响因子的可能性。温度控制也很重要，我们建议在恒温的专用房间内部的运动器材。最后，必须培养和男性和女性分别测量，以避免潜在的生育和性别差异，在运动能力的影响因子。 这种方法的应用潜力是有限的，只有通过研究者的想象力。在前期工作中，我们已经利用这种方法，在三大应用： <ol><li>野生型生物学的各个方面，跨年龄来衡量的耐力锻炼效果。 </li><li> ţO衡量耐力运动对特定的突变表型的影响。 </li><li>遗传因素是必要的筛选，执行运动的好处</li></ol>这些应用程序的每一个囊括了多种具体的可能性。根据我们的初步经验，突变表型往往与尽可能多的锻炼水平不同，因为他们的饮食不同。使用的脊椎动物模型，以便更好地了解饮食，运动和衰老生理之间的关系，也许是最重要的这个协议的普遍应用。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>顿减速机</td><td>格兰杰</td><td> 1LRA6A </td><td></td></tr><tr><td> RACO电气箱</td><td>格兰杰</td><td> 5A052 </td><td></td></tr><tr><td> RACO封面</td><td>格兰杰</td><td> 5A053 </td><td></td></tr><tr><td>库珀Bussmann保险丝</td><td>格兰杰</td><td> 6F043 </td><td></td></tr><tr><td>的Cooper Bussmann保险丝座</td><td>格兰杰</td><td> 1DD33 </td><td></td></tr><tr><td>联赛技术开关</td><td>格兰杰</td><td> 2X464 </td><td></td></tr><tr><td>顿控制，交流/直流调速</td><td>格兰杰</td><td> 4X796 </td><td&gt; </td></tr><tr><td>为窄型塑料瓶flugs </td><td>杰纳西科学</td><td> 49-102 </td><td></td></tr></tbody></table>

endurance training protocol and longitudinal performance assays for drosophila melanogaster

现代医学研究人员面临的最紧迫问题之一是肥胖的涌动的水平，与相 ​​关疾病，如糖 ​​尿病和心血管疾病的1-3也随之增加。这些相关的健康问题研究的一个重要课题涉及的耐力运动作为一个有益的干预作用。 运动训练是一种廉价的，非侵入性干预与一些有益的结果，包括减少体内多余的脂肪4，增加5骨骼肌对胰岛素的敏感性，增加抗发炎和抗氧化反应，改善心肌的收缩能力7。低强度的运动被称为PGC1-α转录激活增加人类和小鼠的线粒体活性和合成，作为一项重要的中间9,10。 despiTE运动作为打击的几个重要的年龄有关的疾病的工具的重要性，广泛的纵向遗传研究已经阻碍了一个短暂的遗传模型物种缺乏耐力训练协议。与果蝇的遗传工具，连同其寿命短和低廉的维修，品种，使其吸引力模型为进一步研究这些基因的机制。考虑到这一点，我们已经开发一种新型的仪器，称为电力塔，大型运动训练果蝇 11。电力塔采用的苍蝇“本能的负面geotaxis行为重复诱发快速登山。每次机升降机，然后下降，苍蝇的平台，苍蝇诱导攀升。苍蝇继续响应只要机器在操作或直到他们变得过于疲劳回应。因此，研究人员可以使用这台机器提供Simultaneous培训大批年龄相匹配的基因完全相同的苍蝇。此外，我们描述了有益的跟踪培训期间飞同伙的纵向进展的相关实验。

Watch this Scientific Journal Video about Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster at JoVE.com

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

我们描述了一个重要的遗传模型物种的耐力训练协议，<em&gt;果蝇</em&gt;，勾勒出几个分析图表改进流动以下培训。

果蝇的耐力训练协议和纵向性能检测

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders ...

endurance-training-protocol-longitudinal-performance-assays-for

Research

JoVE Journal

Biology

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

15.5K 次观看.  University of Michigan Medical School. 我们描述了一个重要的遗传模型物种的耐力训练协议，果蝇，勾勒出几个分析图表改进流动以下培训。

视频: Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

科研

生物学

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit检测癫痫发作癫痫突变果蝇</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

在幼虫的监测心脏功能果蝇生理研究

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

定量比较顺调节元件（CRE）的活动，在转基因果蝇</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

寿命测量<em&gt;果蝇</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

的维持<em&gt;果蝇</em&gt;人口笼

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.